Beads, Buffers and DyesSupport Systems for Flow Cytometry
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Table of ContentsOverview: Beads, Buffers and Dyes 1
1. Cell Preparation 2
Simplify steps . . . . . . . . . . . . . . . . . . . . . . . . 2
Red blood cell lysis . . . . . . . . . . . . . . . . . . . . . 2
Dissociation and detachment . . . . . . . . . . . . . . . . 4
2. Cell Treatments 5
Cell stimulation and protein accumulation . . . . . . . . . 5
3. Blocking 8
Block Fc-mediated binding . . . . . . . . . . . . . . . . . 8
4. Surface and Intracellular Staining 9
Choosing the best buffer system . . . . . . . . . . . . . . 9
Intracellular cytoplasmic staining . . . . . . . . . . . . . .10
Secreted protein: cytokine, chemokine and growth factors .10
Nuclear staining . . . . . . . . . . . . . . . . . . . . . . .11
Fixation buffers . . . . . . . . . . . . . . . . . . . . . . .12
5. Cellular Activity 13
Cell condition . . . . . . . . . . . . . . . . . . . . . . . .13
Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . .16
6. Cell function 17
Calcium sensing reagents . . . . . . . . . . . . . . . . . .17
Cell tracking . . . . . . . . . . . . . . . . . . . . . . . .18
Cell proliferation . . . . . . . . . . . . . . . . . . . . . .19
Cell proliferation dyes . . . . . . . . . . . . . . . . . . .22
Compensation overview. . . . . . . . . . . . . . . . . . .24
Isotype controls . . . . . . . . . . . . . . . . . . . . . . .28
7. Secondary Reagents 29
Selecting the correct secondary antibody . . . . . . . . . .29
Application determines the format . . . . . . . . . . . . .29
Host species and isotype of primary determines the secondary antibody . . . . . . . . . . . . .29
Instrument and fluorochrome/dye chart 32
1
Overview: Beads, Buffers and DyesIs your data affected by lot-to-lot variations and unreliable product quality? Ensuring
experimental conditions are consistent and data is reproducible can be challenging. In
response to your concerns, eBioscience has developed high-quality products that are
validated and optimized in our own buffers and reagents, ensuring reproducible results.
eBioscience is an industry leader and manufacturer of organic dyes and fluorochrome-
conjugated antibodies to both novel and established clones. eFluor® Organic Dyes are a
proprietary line of fluorophores designed by eBioscience for superior optical performance
and detection in applications using laser-based systems, notably flow cytometry.
Many support products have been optimized and simplified by our research and
development team, who are experts in flow cytometry, to reduce error, thereby saving your
laboratory time and money.
Finding the correct products for your research is easy
All eFluor fluorophores are named according to their emission wavelength and are fully
compatible with protein based dyes, such as PE and APC, other organic fluorophores and
semi-conductor quantum dots. These features, combined with a broad biological portfolio,
allow for easy dye selection when optimizing multicolor panel design for flow cytometry.
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2
Cell PreparationSimplify steps
Preparing cells for experimental procedure can be time consuming and laborious. Using
multi-purpose or multi-function reagents saves time and reduces the need for multiple
products. eBioscience provides buffers with the ability to fix/lyse and store cells in one step,
while red blood cell lysis buffers are suitable for multiple species.
Red blood cell lysis
Removal of red blood cells (RBC) is necessary prior to using cell suspensions for flow cytometry
and functional assays. eBioscience provides lysis buffers containing ammonium chloride, which
are suitable for use before or after staining with antibodies in multiple species.
10X RBC Multi-Species Lysis Buffer
This amonium chloride buffer lyses red blood cells with minimal effect on leukocytes, and is
suitable for use with canine, human, mouse, rat and non-human primate samples, such
as peripheral blood and spleen.
1X RBC Lysis Buffer
The 1X Red Blood Cell Lysis Buffer is formulated for convenience, providing optimal
erythrocyte lysis in single-cell suspensions of mouse hematopoietic tissues, such as spleen and
mouse peripheral blood. The buffer lyses red cells with minimal effect on leukocytes when
used as instructed. However, nucleated red cells are not effectively lysed with ammonium
chloride. RBC lysis is not necessary when working with mouse thymus and lymph nodes.
1
Lysis of normal peripheral blood in multiple species using 10X RBC Lysis Buffer (cat. no. 00-4300).
SSC-
A
FSC-A FSC-A
SS
C-A
Human
FSC-A
SS
C-A
FSC-A
SS
C-A
Mouse
Canine
FSC-A
SS
C-A
Rhesus
SSC-
A
FSC-A FSC-A
SS
C-A
Human
FSC-A
SS
C-A
FSC-A
SS
C-A
Mouse
Canine
FSC-A
SS
C-A
Rhesus
MouseHuman
SSC-
A
FSC-A
SSC-
A
FSC-A
FSC-A
SS
C-A
Human
FSC-A
SS
C-A
FSC-A
SS
C-A
Mouse
Canine
FSC-A
SS
C-A
Rhesus
FSC-A
SS
C-A
Human
FSC-A
SS
C-A
FSC-A
SS
C-A
Mouse
Canine
FSC-A
SS
C-A
RhesusCanine Rhesus
One buffer–multiple species
3
Lysis Buffers At-a-Glance
Cat. No. Name Target Feature
00-430010X RBC Lysis Buffer (Multi-species)
n Red blood cellsn Peripheral blood and hematopoietic
tissues, such as spleen
n Optimized for multiple species: Human, mouse, rat & non-human primate
n Use before or after stainingn Excellent choice for whole blood
00-4333 1X RBC Lysis Buffern Red blood cellsn Mouse hematopoietic tissues, such as
spleen and human peripheral bloodn Suitable for mouse and human tissues
00-53331-step Fix/Lyse Solution (10X)
n Human whole bloodn Lyses non-nucleated erythrocytes
n Use in human whole bloodn Lyse & Fix cells in 10 minutesn Allows storage of cellsn Eliminates gradient centrifugationn Compatible with organic dyes, semi-conductor
quantum dots and tandem dyes
1-step Fix/Lyse Buffer �Simple to use
�Suitable for tandem dyes
�Store fixed and stained cells
This solution fixes and lyses RBCs in one step while maintaining the fluorochrome antibody
stained population. It is suitable for lysis of red blood cells after staining peripheral blood
cells with fluorochrome-conjugated antibodies. There is minimal change in FRET efficiency
of stained leukocytes when using tandem dye conjugated antibodies. 1-Step Fix/Lyse Buffer
can be used as a storage buffer with little to no effect on fluorochromes, particularly
tandem dyes.
Normal human peripheral blood cells were stained with Anti-Human CD45 FITC (cat. no. 11-9459), Anti-Human CD3 PerCP-eFluor® 710 (cat. no. 46-0037) and Anti-Human CD19 eFluor® 450 (cat. no. 48-0199) and then incubated with eBioscience, freshly diluted 1-step Fix/Lyse Solution for 15 minutes at room temperature. Cells were spun, washed once in flow stain buffer, then analyzed (left). CD45+ granulocytes (red), monocytes (blue) and lymphocytes (green) can be seen in the forward vs. side scatter plot of total viable cells (right). Analysis of CD45+ lymphocytes.
SSC-
A
FSC-A
CD
3 Pe
rCP-
eFlu
or® 7
10
CD19 eFluor® 450
Fix and lyse in one step
4
Accutase® – Enzyme cell detachment medium
�Excellent for stem cells
Greater cell recovery
�No neutralization
Harmlessly detaches cells from cultureware, allowing for the analysis of cell surface markers;
virus growth assays; quiescence assays by serum starvation; transformation assays by
oncogene transfection; neural crest cell migration assays; cell proliferation; cell haptotaxsis;
tumor cell migration assays; routine cell passage; production scale-up (bioreactor); and flow
cytometry. Accutase is effective on: fibroblasts, keratinocytes, vascular endothelial cells,
hepatocytes, vascular smooth muscle cells, hepatocyte progenitors, primary chick embryo
neuronal cells, bone marrow stem cells, adherent CHO and BHK cells, macrophages, 293
cells, L929 cells, immortalized mouse testicular germ cells, 3T3, Vero, COS, HeLa, NT2,
MG63, M24 and A375 metastatic melanoma, gliomas U251 and D54, HT1080 fibrosarcoma
cells and Sf9 insect cells.
Accumax cell dissociation. Before, cell aggregates; 100x magnification. (left) After, same cells, following Accumax treatment; 100x magnification. (right)
Cell Dissociation & Detachment
Description Application Cat. No.
Accumax - Cell Aggregate Dissociation Medium FA 00-4666
Accutase - Enzyme Cell Detachment Medium FA 00-4555
Dissociation and detachment
Accumax® – Cell aggregate dissociation medium
Effectively dissociates cell clumps in a variety of cell lines, including hybridomas, CHO,
BHK, 293, COS and Sf9 cells for cell counting, viral transfection assays, cell sorting,
flow cytometry and bioreactor scale-up.
5
Cell TreatmentsCell stimulation and protein accumulation
Cell type and experimental procedure are important factors to consider when stimulating
cells. Stimulants can induce a variety of surface proteins, transcription factors, chemokines,
cytokines and growth factors. One commonly used stimulant is a combination of PMA
(Phorbol ester activates protein kinase C) and Ionomycin (calcium ionophore). eBioscience
offers a Cell Stimulation Cocktail (cat. no. 00-4970), which includes both stimulants.
However, for more specific stimulation of T-lymphocytes, CD3 and CD28 antibodies are a
great option. Additionally, lipopolysaccharide (LPS) or CpG are used to induce IL-6, IL-10 or
TNFα production by monocytes, macrophages and dendritic cells. Cytokines, chemokines
and other secreted proteins are generally detected at low levels in resting cells, therefore
they require some sort of stimulation to induce expression for flow cytometric detection.
Once the proteins are expressed, it is necessary to block secretion, preventing movement
through the secretory pathway at the endoplasmic reticulum with Brefeldin A, or at the
Golgi apparatus with Monensin, which allows accumulation to reach detectable levels.
2
Cell Stimulation & Inhibition
Description Application Cat. No.
Cell Stimulation Cocktail (500X) FC, ELISA, FA 00-4970
Cell Stimulation Cocktail (plus protein transport inhibitors) (500X) FC, FA 00-4975
Protein Transport Inhibitor Cocktail (500X) FC, FA 00-4980
Brefeldin A Solution (1000X) FC 00-4506
Monensin Solution (1000X) FC 00-4505
IL-1
7A P
E-eF
luor
® 6
10
CD4 PerCP-eFluor® 710
Rat
IgG
2a P
E-eF
luor
® 6
10
CD4 PerCP-eFluor® 710CD5 PerCP-eFluor 710
Rat
IgG
2a P
E-eF
luor
610
CD4 PerCP-eFluor 710
IL-1
7A P
E-eF
luor
610
CD5 PerCP-eFluor 710
Rat
IgG
2a P
E-eF
luor
610
CD4 PerCP-eFluor 710
IL-1
7A P
E-eF
luor
610
Splenocytes were cultured under Th17-polarizing conditions for 6 days then restimulated for 5 hours with the Cell Stimulation Cocktail (plus protein transport inhibitors) (cat. no. 00-4975). Cells were intracellularly stained with Anti-Mouse CD4 PerCP-eFluor® 710 (cat. no. 46-0042) and Rat IgG2a K Isotype Control PE-eFluor® 610 (cat. no. 61-4321) (left) or Anti-Mouse/Rat IL-17A PE-eFluor® 610 (cat. no. 61-7177) (right) using the Intracellular Fixation & Permeabilization Buffer Set (cat. no. 88-8824) and protocol. Viable cells, as determined by Fixable Viability Dye eFluor® 660 (cat. no. 65-0864), in the lymphocyte gate were used for analysis.
Stimulate cells with a cocktail
Application Key: BA = Bioassay; ELISA; ELISA (c) = ELISA capture; ELISA (d) = ELISA detection; ELISPOT (c) = ELISPOT capture;
ELISPOT (d) = ELISPOT detection; FA = Functional Activity; FC = Flow Cytometry; FF = FlowCytomix™; IC Flow = Intracellular Staining/Flow
Cytometry; ICC = Immunocytochemistry; IHC = Immunohistochemistry; IP = Immunoprecipitation; MIC = Microscopy;
NU = Neutralizing; WB = Western Blot
6
Cytokine Activation, Re-Stimulation & Blocking At-a-Glance
Mouse Cytokine Activation Re-Stimulation Intracellular Block
GM-CSF ConA /IL-2 Anti-CD3 + Anti-CD28 Brefeldin A
IFNγ ConA /IL-2 Anti-CD3 + Anti-CD28 Brefeldin A
IL-1α mIFNγ /LPS - Brefeldin A
IL-2 ConA Anti-CD3 + Anti-CD28 Brefeldin A
IL-4 Th2 polarized PMA + Ionomycin or Cell Stimulation Cocktail Brefeldin A
IL-5 ConA Anti-CD28 Brefeldin A
IL-10 ConA Anti-CD3 + Anti-CD28 Brefeldin A
IL-12/IL-23 (p40) LPS - Brefeldin A
IL-13 Th2 polarized PMA + Ionomycin or Cell Stimulation Cocktail Brefeldin A
IL-22 Th17 polarized PMA + Ionomycin or Cell Stimulation Cocktail Brefeldin A
MCP-1/ CCL2 LPS - Brefeldin A
TNFα ConA Anti-CD3 + Anti-CD28 Brefeldin A
IL-1β LPS - Monensin
IL-6 LPS - Monensin
IL-17A Th17 polarized PMA + Ionomycin or Cell Stimulation Cocktail Monensin
IL-17F Th17 polarized PMA + Ionomycin or Cell Stimulation Cocktail Monensin
IL-21 Th17 polarized PMA + Ionomycin or Cell Stimulation Cocktail Monensin
IL-23 p19 mGM-CSF LPS Monensin
Human Cytokine Activation Re-Stimulation Intracellular Block
IFNγ PMA /Ionomycin - Brefeldin A
IL-1β LPS - Brefeldin A
IL-1RA LPS - Brefeldin A
IL-2 PMA /Ionomycin - Brefeldin A
IL-4 PMA /Ionomycin - Brefeldin A
IL-5 Th2 polarizing cultures PMA + Ionomycin or Cell Stimulation Cocktail Brefeldin A
IL-6 LPS - Brefeldin A
IL-12/ IL-23 (p40) hIFNγ/LPS - Brefeldin A
IL-13 Anti-CD3 + Anti-CD28 PMA + Ionomycin or Cell Stimulation Cocktail Brefeldin A
IL-17A Th17 polarizing cultures PMA + Ionomycin or Cell Stimulation Cocktail Brefeldin A
IL-21 PMA /Ionomycin - Brefeldin A
IL-22 Th17 polarizing cultures PMA + Ionomycin or Cell Stimulation Cocktail Brefeldin A
TNFα PMA /Ionomycin - Brefeldin A
G-CSF LPS - Monensin
GM-CSF PMA /Ionomycin - Monensin
IL-1α LPS - Monensin
IL-9 Th2 polarizing cultures PMA + Ionomycin or Cell Stimulation Cocktail Monensin
IL-10 Th2 polarizing cultures PMA + Ionomycin or Cell Stimulation Cocktail Monensin
IL-23 p19 hGM-CSF+hIL-4 LPS Monensin
MCP-1/ CCL2 LPS - Monensin
RANTES/ CCL5 LPS - Monensin
TNFβ Th1 polarizing cultures PMA + Ionomycin or Cell Stimulation Cocktail Monensin
Annotations: mouse PEC=mouse thioglycolate-elicited peritoneal macrophages; ConA=Concanavalin A; Iono=Ionomycin; LPS=Lipopolysaccharide; PMA=Phorbol Myristate Acetate
Visit ebioscience.com/knowledge-center.htm for more information and a complete listing of products
7
CD28/CD3TCR-mediated activation and co-stimulation
Con ALectin-mediated Mitogenic activation
Ionomycin and PMALeukocyte activation and stimulation
LPS Leukocyte activation
TCR and MHC receptorT cell activation
TCR-MHC/peptideT cell activation
CD28 Receptor
CD3Receptor
LPS
CD14
MHC MHC
TCR TCRSuper-antigen
Ionomycin
PMA
Ca2+Con A
CD28 Receptor
CD3Receptor
LPS
CD14
MHC MHC
TCR TCRSuper-antigen
Ionomycin
PMA
Ca2+Con A
CD28 Receptor
CD3Receptor
LPS
CD14
MHC MHC
TCR TCRSuper-antigen
Ionomycin
PMA
Ca2+Con ACD28 Receptor
CD3Receptor
LPS
CD14
MHC MHC
TCR TCRSuper-antigen
Ionomycin
PMA
Ca2+Con A
CD28 Receptor
CD3Receptor
LPS
CD14
MHC MHC
TCR TCRSuper-antigen
Ionomycin
PMA
Ca2+Con ACD28 Receptor
CD3Receptor
LPS
CD14
MHC MHC
TCR TCRSuper-antigen
Ionomycin
PMA
Ca2+Con A
Activation and stimulation options
T cells require two stimulus for activation with the first signal, which is antigen specific, generated through the T cell receptor (TCR) interacting with the major histocompatibility complex (MHC) on antigen presenting cells (APC). The second interaction occurs between molecules on the APC and T cells. B cells bind antigens with the B cell receptor (BCR), which are then presented on the MHC II molecules, resulting in activation of B cell binding to TCR and the MHC complex.
8
BlockingBlock Fc-mediated binding �Reduce background
�Improve data
High background reduces data quality, making analysis difficult, but by using human Fc
receptor blocking reagents or Anti-Mouse CD16/32, optimal staining and signal-to-noise
ratios can be achieved.
Fc-blocking reagents are used to inhibit non-specific Fc Receptor (FcγR)-mediated binding
of antibodies, thereby allowing optimal staining and signal-to-noise ratio during flow
cytometric analysis. Four different classes of FcγR are expressed at varying levels in multiple
cell lineages, with high expression observed in myeloid and B cells. The biological function
of the FcγR, including initiation of endocytosis, phagocytosis and antigen presentation, is
elicited upon binding of host-immunoglobulin. Binding of FcγR to monoclonal antibodies
varies depending upon isotype.
Human & Mouse Background Blocking At-a-Glance
Name Cat. No. Target Feature
Human Fc Receptor Binding Inhibitor Functional Grade Purified
16-9161
n Four different Fc γ receptor classes: FcγRI (CD64) FcγRII (CD32) FcγRIII (CD16) FcγRIV
n Inhibits non-specific Fcγ receptor mediated binding of antibodies to cell surface Fc receptors
n Does not contain sodium aziden Expressed at varying cell lineages:
myeloid granulocyte B cells
Human Fc Receptor Binding Inhibitor Purified
14-9161 n Contains sodium azide
Anti-Mouse CD16/CD32 Functional Grade Purified
16-0161
n Low affinity receptors for mouse IgG Fc: CD16 (FcγIII Receptor) CD32 (FcγII Receptor)
n Does not contain sodium aziden Expressed by:
B cells monocyte/macrophages NK cells neutrophils
Anti-Mouse CD16/CD32 Purified
14-0161 n Contains sodium azide
3
9
Intracellular Staining Protocols At-a-Glance
Cytoplasmic Staining (Cytokines)
Nuclear Staining (Transcription Factors)
Nuclear & Cytoplasmic* (Cytokines & Transcription Factors)
Legend: IC = Intracellular, Perm = Permeabilization
Surface and Intracellular Staining Choosing the best buffer system
Cell signaling is critical for cell growth, proliferation and repair. Understanding signal
transduction pathways can provide important information on disease development and
progression. Analysis of proteins involved in signaling pathways by flow cytometry requires
consideration as to the location of target proteins. Once known, the appropriate buffer
for surface and intracellular staining, whether it is cytoplasmic or nuclear, can be chosen.
eBioscience buffers have been optimized for nuclear proteins, such as transcription factors,
in addition to cytoplasmic and secreted proteins.
When combining staining of proteins that localize to different regions, choosing the correct
buffer becomes more challenging. For example, in order to obtain optimal staining of a
transcription factor, it is recommended to use the Foxp3/Transcription Factor Staining Buffer Set,
however when including cytokine staining, a dilemma may occur. The following chart provides
a general rule. There may be a decrease in brightness with one buffer system over another,
therefore each antibody should be optimized independently to validate the staining pattern.
4
Stain surface proteins
Fix + permeabilize cells
Wash
Stain proteins
Stain surface proteins
Fix cells
Permeabilize cells
Wash
Stain proteins
Foxp3/Transcription Factor Staining Buffer Set (cat. no. 00-5523)
Kit components:Fixation/Permeabilization Concentrate Fixation/Permeabilization DiluentPermeabilization Buffer (10X)
IC Fixation Permeabilization Buffer Set (cat. no. 88-8823)
Kit components:Brefeldin A IC Fixation Buffer Permeabilization Buffer (10X)
10
Cytoplasmic and Cytokine Buffers At-a-Glance
Name Cat. No. Target Feature
Intracellular Fixation & Permeabilization Buffer plus Brefeldin A
88-8823
n Cytokines
n Cytoplasmic proteins
n Contains Brefeldin A
n Contains cat. no. 00-8222 and 00-8333
Intracellular Fixation & Permeabilization Buffer Set
88-8824 n Contains cat. no. 00-8222 and 00-8333
Intracellular Fixation Buffer 00-8222 n Increases signal-to-noise ratios
Permeabilization Buffer (10x) 00-8333n Permeabilizes cells prior to intracellular
staining
Intracellular cytoplasmic staining
Cytoplasmic staining requires fixation to crosslink protein, thereby preventing cell contents
from escaping once it has been permeabilized. Intracellular fixation and permeabilization
buffers are ideal for optimal detection of cytoplasmic proteins, cytokines and other secreted
proteins. The eBioscience Intracellular Fixation & Permeabilization Buffer Set is designed
for use when staining proteins, such as adaptor proteins β-catenin, actin and tubulin,
in addition to receptor proteins in which the antibody recognizes a cytoplasmic version
(CD152 (CTLA-4)) or cytoplasmic domain.
Secreted protein: cytokine, chemokine and growth factors
Proteins that go through the secretory pathway, such as cytokines, chemokines and growth
factors also require fixation and permeabilization in addition to protein accumulation for
optimal detection in flow cytometry analysis. This can be accomplished by blocking protein
movement to different organelles and secretion through the use of transport inhibitors.
Cytokine
DNA
Extracellular
Cytoplasm
Nucleus
Cytokine
DNA
Extracellular
Cytoplasm
Nucleus
Cytokine
DNA
Extracellular
Cytoplasm
Nucleus
Intracellular Staining
Cytokine
DNA
Extracellular
Cytoplasm
Nucleus
Cytokine
DNA
Extracellular
Cytoplasm
Nucleus
Cytokine
DNA
Extracellular
Cytoplasm
Nucleus
Nuclear Staining
11
Nuclear Staining Buffers At-a-Glance
Description Cat. No. Target Feature
Foxp3/Transcription Factor Staining Buffer Set 00-5523
n Nuclear factors
n Transcription factors
n Cytosolic proteins
n Secreted proteins
n Works with all transcription factors and most nuclear proteins
n Contains cat. no. 00-5123, 00-5223 & 00-8333
Fixation/Permeabilization Concentrate 00-5123 n Transcription factors
n Cytokines
n Works with all transcription factors and most nuclear proteins
Fixation/Permeabilization Diluent 00-5223
Foxp3 Fixation/Permeabilization Concentrate and Diluent
00-5521
n Nuclear factors
n Transcription factors
n Cytosolic proteins
n Secreted proteins
n Works with all transcription factors and most nuclear proteins
n Contains cat. no. 00-5123 & 00-5223
Staining Buffer At-a-Glance
Description Cat. No. Target Feature
Flow Cytometry Staining Buffer 00-4222
n Tissue culture cell preparation
n Lymphoid tissue cell preparation
n Non-lymphoid tissue cell preparation
n Isolation of PBMC from whole blood
n Optimized for flow cytometry
n Ideal pH
Nuclear staining
Transcription factors are key groups of molecules involved in regulating gene expression by
modulating the synthesis of messenger RNA. Cellular functions, such as cell proliferation,
differentiation and apoptosis, are mediated by transcription factors that are either
up-regulated or blocked, causing inflammatory responses and tumorigenesis. Foxp3
is considered the master regulator of T regulatory cells (Treg). Induction of the Foxp3
gene in normal naïve T cells converts them to Treg-like cells with in vivo and in vitro
suppressive function, indicating that Foxp3 plays a key role in controlling expression of
critical suppression-mediating molecules. Elucidation of the molecular targets of Foxp3
will be fundamental to a complete understanding of the suppressive functions of Tregs.
eBioscience, having mapped the epitopes of Foxp3 antibodies, is an industry leader in
providing tools for the identification of Tregs. The Foxp3/Transcription Factor Staining Buffer
Set, although originally developed for Foxp3 staining, has been optimized for use with
nuclear factors, cytosolic proteins, secreted proteins and transcription factors in addition
to cytokines. These include Eomes, T-bet, Gata-3, IRF4, phospho-H2Ax, Rorγ(t), Egr2,
Ki-67 and Sox2. This buffer can also be used to stain many secreted proteins (IL-17A and
Granzyme B).
12
Fixation buffers
Fixation buffers for flow cytometry are generally used after surface staining and as an
early step during intracellular staining protocols. However, fixation buffers are also a great
reagent on occasions when you cannot gain access to the cytometer for hours or days but
need to preserve both the cells and staining. Intracellular Fixation Buffer (cat. no. 00-8222)
and 1-step Fix/Lyse Buffer (cat. no. 00-5333) for flow cytometry are manufactured with the
highest quality materials, allowing for safe cell storage when it is impossible to run samples
immediately. eBioscience fixatives have minimal impact on tandem dye brightness
or compensation values when compared to freshly stained live cells.
IC Fixation Buffer for 3 days (100 μL cells+ 100 μL IC Fix Buffer) Compensation: 5%
1-Step Fix/Lyse for 3 days (100 μL cells + 2 mL Fix/Lyse Buffer) Compensation: 5%
Live cells Compensation: 4.5%
PE-Cy7 APC-eFluor® 780
Tandem dyes can be stored in eBioscience fixatives with virtually no change in compensation/FRET efficiency when compared to freshly stained and analyzed cells. The data shows minimal impact to tandem brightness and compensation of mouse CD4 (RM4-5) after being stored for periods of three days in 1-step Fix/Lyse or solution.
1. Finish standard staining protocol.
2. Wash and decant cells in 100 μL staining buffer.
3. Add eBioscience Fixative: 100 μL of IC Fixation Buffer
or 2 mL 1-step Fix/Lyse Solution.
4. Store at 2-8°C, in the dark, for up to 3 days.
5. Wash and analyze using standard protocol when ready.
Stain
Wash
Fix & Store
Wash and Analyze
Simple Storage Protocol
eBioscience fixatives have minimal impact on tandem brightness or compensation
13
Cellular ActivityCell condition
Excluding dead cells from data is recommended for all staining protocols to ensure accurate
data. Dead cells can be “sticky”, resulting in non-specific binding, which causes high
background staining and false positives. Gating based on forward and side scatter
(FSC/SSC) is not a reliable method for distinguishing dead cells. Viability dyes ensure dead
cells are removed from analysis, thereby reducing non-specific binding and background in
addition to better peak separation. eBioscience offers several options for ensuring only live
cells are analyzed, such as DNA-intercalating dyes, enzymatically sensitive dyes and fixable
viability dyes.
eFluor® Fixable Viability Dyes
Ready-to-use format
Excludes dead cells easily
Improves data quality
eBioscience eFluor® Fixable Viability Dyes (FVD) penetrate compromised membranes, irreversibly
labeling dead cells from all species prior to fixation and permeabilization. Dead cell populations
have high fluorescent intensity, allowing for easy exclusion, thereby improving data quality.
Fixable Viability Dyes eFluor® 455UV, eFluor® 450, eFluor® 506, eFluor® 520, eFluor® 660
and eFluor® 780 are permanent dyes suitable for UV, violet, blue and red lasers that will not
wash out of cells. Unlike 7-AAD and propidium iodide, cells labeled with Fixable Viability Dyes
can be cryopreserved or fixed, permeabilized and stained for intracellular antigens without
losing staining intensity of the dead cells.
5
The importance of viability dyes
Bottom row: Total cells were subgated on viable cells, based on FVD eFluor 520, then analyzed for CD4 and CD8.
CD
8 A
PC
CD4 PEFixa
ble
Via
bilit
y D
ye e
Fluo
r® 5
20
Forward Scatter
CD
8 A
PC
CD4 PE
Side
Sca
tter
Forward Scatter
Mouse splenocytes were stimulated with ConA. for 4 days
Top row: Total cells analyzed for CD4 and CD8, based on a forward and side scatter gate.
14
7-AAD Viability Dye and Propidium Iodide (PI) Staining Solution
�Ready-to-use format
�Dead cell discrimination
7-AAD Viability Dye and Propidium Iodide (PI) Staining Solution mark non-viable cells by
intercalating with the DNA of dead cells in a concentration-dependent manner. The nucleic
acid of viable cells will not be accessible to the dye, thereby preventing staining. They are not
recommended for viability gating if the cells will be fixed. 7-AAD and PI may also be used for
flow cytometric analysis of the cell cycle. PI binds to the double stranded DNA of dead cells,
but is excluded from cells with intact plasma membranes.
Dead Cell Staining At-a-Glance
Description Format Utility
Via
bili
ty
Ap
plic
atio
n
Exci
tati
on
Emis
sio
n
Cat
. No
.
UV Laser
Fixable Viability Dye eFluor® 455UV
Solution/ Ready-to-use
Irreversibly labels dead cells prior to cryopreservation, fixation and/or permeabilization procedures. Cells can be washed without loss of staining on dead cells, allowing for exclusion during analysis
Dead Cells
FC 350 nm 455 nm 65-0868
Violet Laser
Fixable Viability Dye eFluor® 450 Solution/
Ready-to-use
Irreversibly labels dead cells prior to cryopreservation, fixation and/or permeabilization procedures. Cells can be washed without loss of staining on dead cells, allowing for exclusion during analysis
Dead Cells
FC405 nm 450 nm 65-0863
Fixable Viability Dye eFluor® 506
405 nm 506 nm 65-0866
Blue Laser
Fixable Viability Dye eFluor® 520
Solution/ Ready-to-use
Irreversibly labels dead cells prior to cryopreservation, fixation and/or permeabilization procedures. Cells can be washed without loss of staining on dead cells, allowing for exclusion during analysis
Dead cells
FC 488 nm
522 nm 65-0866
Propidium Iodide (PI)
Binds to double stranded nucleic acid by intercalating between basepairs. Excluded from cells with intact plasma membranes.
Use FL3 for viability exclusion. Analyze in FL2 when used as a counterstain for FITC Annexin V.
617 nm 00-6990
7-AAD Viability Staining Solution
Use in place of PI (propidium iodide) or in combination with PE (phycoerythrin) and FITC (fluorescein isothiocyanate) conjugated antibodies in multi-color analysis. Minimal spectral overlap between these emissions.
670 nm 00-6993
Red Laser
Fixable Viability Dye eFluor® 660 Solution/
Ready-to-use
Irreversibly labels dead cells prior to cryopreservation, fixation and/or permeabilization procedures. Cells can be washed without loss of staining on dead cells, allowing for exclusion during analysis
Dead Cells
FC633 nm 660 nm 65-0864
Fixable Viability Dye eFluor® 780
633 nm 780 nm 65-0865
Multi-Laser Pack
Fixable Viability Dye eFluor® 506/780
Solution/ Ready-to-use
Irreversibly labels dead cells prior to cryopreservation, fixation and/or permeabilization procedures. Cells can be washed without loss of staining on dead cells, allowing for exclusion during analysis
Dead Cells
FC405/633 nm
506/780 nm 65-2860
15
Calcein AM, Calcein Blue AM, Calcein Violet 450 AM
Calcein labeling dyes cross the cell membrane easily, selectively labeling live cells for analysis
by flow cytometry or fluorescent microscopy; however apoptotic and dead cells with
compromised cell membranes do not retain Calcein.
Calcein dyes are nonfluorescent until they cross the cell membrane of viable cells and are
enzymatically processed by intracellular esterases to their fluorescent, membrane non-
permeable form. Dead cells do not have intact cell membranes and cannot retain the
cleaved Calcein dyes, nor do they have active esterases to cleave the Calceins to their
fluorescent forms. Co-staining with Annexin V or 7-AAD is recommended to allow the
greatest resolution between live and dead/apoptotic cells.
Live Cell Labeling At-a-Glance
Description Format Utility
Via
bili
ty
Ap
plic
atio
n
Exci
tati
on
Emis
sio
n
Cat
. No
.
UV Laser
Calcein Blue AM Lyophilized
Membrane permeable live cell labeling dye
Co-stain with Annexin V or 7-AAD for greatest resolution between live and dead/apoptotic cells
Live cells
FC/ Microscopy
360 nm 445 nm 65-0855
Violet Laser
Calcein Violet 450 AM Lyophilized
Membrane permeable live cell labeling dye
Co-stain with Annexin V or 7-AAD for greatest resolution between live and dead/apoptotic cells
Live cells
FC/ Microscopy
408 nm 450 nm 65-0854
Blue Laser
Calcein AM (Ultra Pure)
Lyophilized
Membrane permeable live cell labeling dye
Co-stain with Annexin V or 7-AAD for greatest resolution between live and dead/apoptotic cells
For improved resolution of live and dead/apoptotic cells using single color analysis, Calcein Blue AM or Calcein Violet 450 AM are recommended
Live cells
FC/ Microscopy
495 nm 515 nm 65-0853
Calcein Violet 450 AM excited by the violet laser
Cal
cein
Vio
let
450
AM
Annexin V APC
% o
f M
ax
Calcein Violet 450 AM
Application Key: BA = Bioassay; ELISA; ELISA (c) = ELISA capture; ELISA (d) = ELISA detection; ELISPOT (c) = ELISPOT capture; ELISPOT (d) = ELISPOT detection;
FA = Functional Activity; FC = Flow Cytometry; FF = FlowCytomix™; IC Flow = Intracellular Staining/Flow Cytometry; ICC = Immunocytochemistry; IHC = Immunohistochemistry;
IP = Immunoprecipitation; MIC = Microscopy; NU = Neutralizing; WB = Western Blot
Balb/c thymocytes were stained with 1 µM Calcein Violet 450 AM (cat. no. 65-0854) for 30 minutes at room temperature (left). Thymocytes were kept on ice overnight (shaded histogram) or cultured overnight at 37°C without (purple) or with (blue) 1 µM dexamethasone. Thymocytes cultured overnight without dexamethasone were also stained with Annexin V-APC (cat. no. 88-8007) allowing further discrimination between live and dead cells (right). Total cells were used for analysis.
16
Apoptosis
In early-stage apoptosis, the plasma membrane excludes viability dyes such as propidium
iodide (PI), 7-AAD, or fixable viability dyes (FVD) such as FVD eFluor® 660 or eFluor® 780.
These cells will stain with Annexin V due to phosphotidlyserine (PS) present in the inner
plasma membrane moving to the outer membrane, but not a viability dye, distinguishing
cells in early apoptosis. However, in late stage apoptosis, the cell membrane loses integrity,
thereby allowing the DNA intercalating of FVD dyes to enter the interior of the cell. A viability
dye can be used to resolve these late-stage apoptotic and necrotic cells (Annexin V, viability
dye-positive) from the early-stage apoptotic cells (Annexin V positive, viability dye-negative).
Annexin V
Annexin V is a family of calcium-dependent phospholipid-binding proteins that preferentially
binds to PS, thereby identifying apoptotic cells. Under normal physiologic conditions, PS
is predominantly located in the inner leaflet of the plasma membrane. Upon initiation
of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is
translocated to the extracellular membrane leaflet, marking cells as targets for phagocytosis.
Once on the outer surface of the membrane, PS can be detected by fluorescently labeled
Annexin V in a calcium-dependent manner. eBioscience offers many fluorchrome-
conjugated formats of Annexin V.
JC-1
JC-1 is a membrane permeable dye widely used for determining loss of mitochondrial
membrane potential associated with apoptosis or cell stress in flow cytometry and
fluorescent microscopy. The dye selectively enters the mitochondria where it reversibly
changes color as membrane potentials increase (over values of about 80-100 mV).
This property is due to the reversible formation of JC-1 aggregates upon membrane
polarization which causes shifts in emitted light from 530 nm (i.e., emission of JC-1
monomeric form) to 590 nm (i.e., emission of J-aggregate) when excited at 488 nm.
Both colors can be detected using filters for FITC and PE, respectively. JC-1 is both
qualitative, with respect to shift from green to orange fluorescence emission, and
quantitative, as measured by fluorescence intensity.
Detecting Cell Death
Description Application Cat. No.
Annexin V Apoptosis Detection Kit eFluor® 450 FC 88-8006
Annexin V Apoptosis Detection Kit FITC FC 88-8005
Annexin V-FITC Apoptosis Detection Kit FC BMS500FI
Annexin V-FITC Apoptosis Detection Kit FC BMS500FICE
Annexin V PE Apoptosis Detection Kit PE FC 88-8102
Annexin V Apoptosis Detection Set PE-Cy7 FC 88-8103
Annexin V Apoptosis Detection Kit APC FC 88-8007
Annexin V Apoptosis Detection Kit PerCP-eFluor® 710 FC 88-8008
Annexin V-Biotin Apoptosis Detection Kit FC BMS500BT
Binding Buffer for Annexin V FC BMS500BB
JC-1 Mitochondrial Membrane Potential Dye FC, IHC 65-0851
17
Cell functionCalcium sensing reagents
Calcium Sensing Dye eFluor® 514
Membrane-permeable dyes, such as Calcium sensing dye eFluor® 514 can be used to
monitor changes in intracellular free calcium concentrations in the cell using fluorescence
microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers.
Calcium Sensor Dye eFluor® 514 enters the cell with an acetoxymethyl (AM) ester group that
is cleaved by cellular esterases yielding a membrane-impermeable dye fluorescing at ~520
nm when excited with the 488 nm laser. Calcium Sensor Dye eFluor® 514, like Fluo-3 and
Fluo-4, is a commonly used dye among the visible light-excitable calcium indicators, but with
increased cellular uptake and brightness, even at room temperature.
Indo-1 AM Calcium Sensor Dye
This membrane-permeable dye is used for determining changes in calcium concentrations
within the cell using fluorescence microscopy, flow cytometry, fluorescence spectroscopy
and fluorescence microplate readers. Once Indo-1 enters the cell, esterases cleave the AM
ester group, yielding a membrane-impermeable dye with a peak excitation wavelength of
346 nm. Unbound Indo-1 has a peak emission at 485 nm. Upon binding calcium, the peak
emission shifts down to 410 nm.
Flow cytometry enables Indo-1 AM to be measured over time and can be represented as a
ratio of the two emission wavelengths. To optimize the ratio between the two emissions,
unbound Indo-1 fluorescence should be collected using a filter above 485 nm (525 nm
is a good option), while bound Indo-1 fluorescence should be collected using a filter
below 400 nm. Because the emission profile of Indo-1 is broad, multicolor flow analysis
using fluorochrome off the violet laser is not possible; however fluorochrome-conjugated
antibodies utilizing the 488 nm or 633 nm laser lines are compatible with Indo-1.
Cell Monitoring
Description Application Cat. No
Calcium Sensor Dye eFluor® 514 FC, IHC 65-0859
Indo-1 AM Calcium Sensor Dye FC, IHC 65-0856
Indo-1 AM Calcium Sensor Dye (UltraPure Grade) FC, IHC 65-0857
7
Application Key: BA = Bioassay; ELISA; ELISA (c) = ELISA capture; ELISA (d) = ELISA detection; ELISPOT (c) = ELISPOT capture;
ELISPOT (d) = ELISPOT detection; FA = Functional Activity; FC = Flow Cytometry; FF = FlowCytomix™; IC Flow = Intracellular Staining/Flow
Cytometry; ICC = Immunocytochemistry; IHC = Immunohistochemistry; IP = Immunoprecipitation; MIC = Microscopy;
NU = Neutralizing; WB = Western Blot
18
Cell Tracking Dyes
Description Excitation Emission Application Cat. No.
Violet Laser
CellVue® Lavender Cell Labeling Kit 420 nm 461 nm FC, ICC, IHC, FA 88-0873
Blue Laser
CellVue® Jade Cell Labeling Kit 478 nm 508 nm FC, ICC, IHC, FA 88-0876
Red Laser
CellVue® Maroon Cell Labeling Kit 647 nm 667 nm FC, ICC, IHC, FA 88-0870
CellVue® Plum Cell Labeling Kit 652 nm 671 nm FC, ICC, IHC, FA 88-0871
CellVue® Burgundy Cell Labeling Kit 683 nm 707 nm FC, ICC, IHC, FA 88-0872
CellVue® NIR780 Cell Labeling Kit 633 nm 776 nm FC, ICC, IHC, FA 88-0875
CellVue® NIR815 Cell Labeling Kit 786 nm 814 nm ICC, IHC, FA 88-0874
CellVue® Diluent C FC, IHC 00-4501
Cell tracking
CellVue dyes offer a stable method to rapidly label the cell membrane of live cells with
lipophilic dyes suitable for flow cytometry and microscopy.
CyTRAK Orange™
CyTRAK Orange™ is an anthraquinone dye with high affinity for double-stranded DNA. It is a
membrane-permeable dye that can label live or fixed/dead cells. In flow cytometry, it can be
used to distinguish nucleated and non-nucleated cells. In fluorescent microscopy, it can be used
to identify and discriminate the nucleus and cytoplasm without the need for a second dye, due
to its high intensity staining of the nucleus and low intensity staining of the cytoplasm. CyTRAK
Orange is optimally excited from 488 to 550 nm with a peak emission of 610 nm.
DRAQ5™
DRAQ5™ is an anthraquinone dye with high affinity for double-stranded DNA. It is a
membrane-permeable dye that can label live or fixed/dead cells. In flow cytometry, this
dye can be used to distinguish nucleated and non-nucleated cells. DRAQ5 can also be
used to report nuclear DNA content for ploidy and cell cycle analysis because it binds DNA
stoichiometrically. In fluorescent microscopy, it can be used as a nuclear counterstain.
DRAQ5 can be excited from 488-647 nm with a peak emission of 670 nm.
CyTRAK Orange™ U2-OS human osteosarcoma cells, counterstained with CyTRAK Orange™ (courtesy of Biostatus).
Membrane Permeable Dyes
Description Application Cat. No
CyTRAK Orange™ FC, ICC, IHC, FA 65-0881
DRAQ5™ FC, ICC, IHC, FA 65-0880
DRAQ5™ Fixed and permeabilized MDCK cells stained with 10 nM DRAQ5™ nuclear stain (cat. no. 65-0880) (red), 20X.
Application Key: BA = Bioassay; ELISA; ELISA (c) = ELISA capture; ELISA (d) = ELISA detection; ELISPOT (c) = ELISPOT capture;
ELISPOT (d) = ELISPOT detection; FA = Functional Activity; FC = Flow Cytometry; FF = FlowCytomix™; IC Flow = Intracellular Staining/Flow
Cytometry; ICC = Immunocytochemistry; IHC = Immunohistochemistry; IP = Immunoprecipitation; MIC = Microscopy;
NU = Neutralizing; WB = Western Blot
19
Cell proliferation
The study of cell proliferation is important in the understanding of uncontrolled cell growth
as a result of cancer, in addition to cell development, regulation and differentiation.
Understanding the effects of gene addition or deletion and chemical additives on cells can
be observed with proliferation assays.
Methods to Evaluate Cell Proliferation
BrdU Ki-67 PCNA Proliferation Dyes
Measures cells in S-phase only Measures proliferating cells at any cell cycle stage except G0
Measures S-phase but also includes late G1 phase
Measures generational proliferation
Pulse-labeling common to avoid cytotoxicity
BrdU is a subset of Ki-67 positive cells
Data supports IHC applications Not as robust for flow cytometry
Long-term labeling assay. Does not require fixation
In long-term culture, BrdU can be pulse-labeled and washed out
Dividing cells will not incorporate BrdU so toxicity is diluted
Ki-67 and BrdU are used together in both IHC and flow cytometry
Cannot distinguish cell cycle phases of daughter cells
Cell cycle analysis
G0 phase: Resting cells have zero growth
G1 phase: Enzyme synthesis is required for DNA replication
S-phase: DNA replication producing two identical sets of chromosomes
G2 phase: Protein synthesis occurs
Mitosis: The nucleus and cell divide
G0 phase
S-phase
G1 phaseG2 phase
Mitosis
20
BrdU
�Fast Protocol
�1-step proprietary buffer
BrdU is a synthetic analog for thymidine, integrated into DNA during S-phase. BrdU
incorporation is measured using an Anti-BrdU antibody showing at least one round
of S-phase has been completed.
3H- thymidine-/MTT assays are a sensitive and accurate way to measure overall
proliferation, although information is unavailable as to which cells have gone through
S-phase. A specific instrument is required for reading results, while BrdU is evaluated
using a standard flow cytometer.
The eBioscience BrdU Proliferation Assay is simple and easy-to-use with few steps,
faster results and a room temperature fix/perm protocol. The BrdU assay is compatible with
staining both surface and intracellular targets. The BrdU Staining Buffer Set is optimized for
use in the proliferation assay, enabling improved BrdU staining and consistency.
Similar molecular structures – identical results
H C3 HH HH
HOHO
Br
O
O OO
O
O
OH
Thymidine BrdU
OH
NN NN
Thymidine BrdU
Flow cytometry BrdU staining: Anti-Mouse CD3 and CD28 stimulated mouse splenocytes, either unlabled (left) or labeled with BrdU (right) were intracellularly stained with Anti-Mouse CD4 eFluor® 450 (cat. no. 48-0041) and Anti-BrdU FITC using the BrdU staining kit.
Q144.9%
Q220.3%
Q37.92%
Q426.9%
CD4 eFluor 450
BrdU
FIT
C
Q10.265%
Q20.493%
Q326.0%
Q473.3%
CD4 eFluor 450
BrdU
FIT
C
Eomes PE
% o
f Max
Population Name
Q1: Comp-eFluor 450-A- , Comp-FITC-A+Q2: Comp-eFluor 450-A+ , Comp-FITC-A+Q3: Comp-eFluor 450-A+ , Comp-FITC-A-Q4: Comp-eFluor 450-A- , Comp-FITC-A-
BrdU
FIT
C
CD4 eFluor® 450
unlabeled Q144.9%
Q220.3%
Q37.92%
Q426.9%
CD4 eFluor 450
BrdU
FIT
C
Q10.265%
Q20.493%
Q326.0%
Q473.3%
CD4 eFluor 450
BrdU
FIT
C
Eomes PE
% o
f Max
Population Name
Q1: Comp-eFluor 450-A- , Comp-FITC-A+Q2: Comp-eFluor 450-A+ , Comp-FITC-A+Q3: Comp-eFluor 450-A+ , Comp-FITC-A-Q4: Comp-eFluor 450-A- , Comp-FITC-A-
BrdU
FIT
C
CD4 eFluor® 450
Proliferating cells
BrdU labeled
BrdU measures S-phase proliferation
21
BrdU Proliferation Staining Kits
Description Cat. No.
eFluor® 450 8848-6600-42
FITC 8811-6600-42
PE 8812-6600-42
PerCP-eFluor® 710 8846-6600-42
APC 8817-6600-42
BrdU Staining Buffer Set Cat. No.
BrdU optimized buffers for use with proliferation assays 00-5525-00
Comparative Workflow for BrdU Staining
Competitor Protocol eBioscience Protocol
1. Label cells with BrdU
2. Surface stain (optional)
3. Fix/Perm for 30 minutes
at room temperature
4. Incubate with perm buffer
for 10 minutes on ice
5. Fix cells for 5 minutes at room temperature
6. Treat cells with DNase I for 1 hour at 37°C
7. Stain for BrdU (and other intracellular
antigens) for 20-30 minutes at 2-8°C
8. Analyze samples
1. Label cells with BrdU
2. Surface stain (optional)
3. Fix/Perm in BrdU Staining Buffer for
15 minutes at room temperature*
4. Treat cells with DNase I for 1 hour at 37°C
5. Stain for BrdU (and other cell surface/
intracellular antigens) for 20-30 minutes
at 2-8°C
6. Analyze samples
* Stopping point (up to 16 hours tested)
22
Cell proliferation dyes
The Cell Proliferation Dyes (CPD) eFluor® 670, CPD eFluor® 450 and 5-(and 6)-
Carboxyfluorescein diacetate succinimidyl ester (CFSE) are fluorescent dyes that can be
used to monitor individual cell divisions. Cells labeled with CPD or CFSE may be fixed and
permeabilized for analysis of intracellular targets using standard formaldehyde-containing
fixatives and saponin-based permeabilization buffers, such as the Foxp3/Transcription Factor
Staining Buffer Set (cat. no. 00-5523) or the Intracellular Fixation Buffer (cat. no. 00-8222)
and Permeabilization Buffer (cat. no. 00-8222).
CPD bind to cellular proteins containing primary amines. While cells divide, the dye is
equally distributed between daughter cells, enabling measurement of successive halving of
fluorescence intensity. Between six and eight generations may be visualized, depending on
which dye is used. CPD may also be used for long-term tracking of non-dividing, labeled cells.
CFSE readily crosses intact cell membranes reacting with primary amines, crosslinking
the dye to intracellular proteins. Cell division is measured as successive halving of CFSE
fluorescence intensity, for up to seven generations. CFSE may also be used for long-term
tracking of non-dividing, labeled cells.
Detect cell division in labeled cells
Parent Cell
1st division
2nd division
3rd division
Cell division results in equal distribution of dye between daughter cells. Fluorescence intensity of progeny cells is half that of parent cell
Fluorescence
Cel
l num
ber
Parent123
23
eFluor® Cell Proliferation Dyes
�Excellent dye distibution
�Compatible with GFP
�Lyophilized
eFluor® Cell Proliferation Dyes are membrane permeable fluorescent dyes bound to cellular
proteins containing primary amines, which can be used in vitro or in vivo and visualized
for up to 7 generations. Fluorescent dye binds to cellular proteins and is evenly distributed
between the daughter cells as they divide. eFluor Cell Proliferation Dyes are supplied in a
lyophilized format, that once reconstituted are stable for 6 months when protected from
light and stored at -20°C.
Left: Mouse splenocytes were labeled with 10 uM Cell Proliferation Dye eFluor® 450 and cultured for 3 days with (blue histogram) or without (purple histogram) ConA. Cells were stained with Anti-Mouse CD4 PE (cat. no. 12-0042) and 7-AAD (cat. no. 00-6993). Viable CD4+ cells were used for analysis.
Right: Splenocytes from Thy1.1 mice were labeled with 10 uM Cell Proliferation Dye eFluor® 450 and then injected into C57Bl/6 mice (purple histogram) or B6D2F1 mice (blue histogram). Splenocytes from the C57Bl/6 and B6D2F1 mice were collected 72 hours after injection of the labeled cells. Cells were stained with Anti-Mouse CD4 APC (cat. no. 17-0042), Anti-Mouse/Rat CD90.1 (Thy1.1) PE (cat. no. 12-0900) and Fixable Viability Dye eFluor® 780 (cat. no. 65-0865). Viable Thy1.1+CD4+ cells were used for analysis. Thy1.1-CD4+ host cells from the B6D2F1 mice, that are unlabeled with the Cell Proliferation Dye, are shown in gray.
C e l l P r o l i f e r a t i o n D y e e F l u o r 4 5 0
Cou
ntC
ount
Cell Proliferation Dye eFluor® 450
In Vitro
C e l l P r o l i f e r a t i o n D y e e F l u o r 4 5 0
Cou
ntC
ount
Cell Proliferation Dye eFluor® 450
In Vivo
Tracking daughter cells
Cell Proliferation Dyes
Description Excitation Emission Size Cat. No.
Violet Laser
Cell Proliferation Dye eFluor® 450 409 nm 450 nm 500 ug 65-0842-85
4 x 500 ug 65-0842-90
Blue Laser
CFSE 488 nm 521 nm 5 x 500 ug 65-0850-84
Red Laser
Cell Proliferation Dye eFluor® 670 633 nm 670 nm 500 ug 65-0840-85
4 x 500 ug 65-0840-90
24
8 Performance ControlsUsing controls in your research ensures data accuracy and provides reassurance to review
committees, resulting in less chance of paper rejection, faster approval times and greater
opportunities to publish. Every laboratory should perform compensation with beads or cells
to ensure accurate mean fluorescent intensity is attained, while isotype controls help confirm
the specificity of antibody binding.
Compensation overview
Compensation is the process of subtracting spectral spillover from one fluorochrome
out of the detector of another, and is generally conducted with cells or beads.
While using cells for compensation can be a good choice, beads are a great alternative
when the cell source is limited or the antigen of interest is expressed at low levels or
on a rare subpopulation of cells. UltraComp and OneComp eBeads® ensure accurate
compensation with a guaranteed negative and positive bead population.
530/30
500 550 600 650 700 750
585/42
Wavelength (nm)
650/LP
Emission spectra of fluorochrome overlap
eFlu
or® 6
50N
C
Comp-eFluor® 605NC
eFlu
or® 6
50N
C
CD8 eFluor® 605NC
CellsBeads Using cells for compensation can result in difficulty identifying positive and negative cells.
25
IL-13 staining on cells
n�Broad distribution
n�Few positive events
n��Difficult to identify positive population
PerC
P-eF
luor
® 7
10
IL-13 PE
PerC
P-eF
luor
® 7
10
IL-13 PE
Cou
nt
IL-13 PE
IL-13 staining on OneComp eBeads
n�Discrete distribution
n�50% positive
n��Easy to identify positive population
Take the guesswork out of compensation
Cou
ntIL-13 PE
Good compensation requires similar MFI
Lysed whole blood was stained with CD4 (clone SK3) PE and compensated for spectral spillover of eFluor® 605NC. Performance was compared with a competitor's compensation bead in addition to UltraComp eBeads® which have been optimized for the violet laser.
Competitor's compensation beads on the violet laser
eFluor® 605NC MFI: 69
eFluor® 605NC MFI: 280
PE
UltraComp eBeads optimized for the violet laser
eFluor® 605NC MFI: 67
eFluor® 605NC MFI: 72
PE
Mean Fluorescent Intensity (MFI)
Mean Fluorescent Intensity (MFI) is used in compensation to measure the shift in fluorescent
intensity of a population of cells. Fluorescent intensity is determined by comparing negatively
stained controls in addition to an antibody. A dim positive stain is slightly brighter than the
negative control, whereas the bright stain is generally two logs brighter.
26
Comparing Compensation Beads
Description Species CompatibilityChain
RecognitionFeatures
Mo
use
Ig
Rat
Ig
Ham
ster
Ig
Rab
bit
Ig*
Kap
pa
Li
gh
t C
hai
n
Lam
bd
a
Lig
ht
Ch
ain
On
e D
rop
On
e V
ial
All
cel
l siz
es
Vio
let
Lase
r
UltraComp eBeads® 01-2222 n n n n n n n n n n
OneComp eBeads® 01-1111 n n n n n n n n n
Competitor X Anti-Mouse Ig, k n n Competitor X Anti-Rat Ig, k n n Competitor X Anti-Rat/Hamster Ig, k n n n Competitor X Plus Anti-Mouse Ig, k n n Competitor X Plus Anti-Rat Ig, k n n Competitor Z Anti-Mouse Bead Kit n n n
Competitor Z Anti-Rat/Hamster Bead Kit n n n n
*Rabbit antibodies stain the beads very dimly, but may be useful for some experiments.
OneComp eBeads®
Immunoglobulins PE: Staining of OneComp eBeads with a variety of PE-conjugated monoclonal antibodies.
Mouse IgG1, (CD4)
Mouse IgG2a, (CD3)
Mouse IgG2b, (CD4)
Mouse IgG3, (CD3)
Mouse IgM, (TRA-1-60)
Rat IgG1, (CD25)
Rat IgG2a, (CD4)
Rat IgG2b, (CD45)
Rat IgG2c, (Ly-6D)
Rat IgM, (CD49b)
Rat IgG2a, (CD96)
Golden Syrian Hamster IgG (CD3e)
Armenian Hamster IgG (CD3e)
UltraComp eBeads®
Immunoglobulins eFluor® 450: Staining of UltraComp eBeads with a variety of eFluor® 450 conjugated monoclonal antibodies.
Mouse IgG1, (CD4)
Mouse IgG2a, (CD3)
Mouse IgG2b, (CD4)
Mouse IgG3, (CD3)
Mouse IgM, (CD15)
Rat IgG1, (CD25)
Rat IgG2a, (CD4)
Rat IgG2b, (CD45)
Rat IgG2c, (Ly-6D)
Rat IgM, (CD49b)
Rat IgG2a, (93)
Golden Syrian Hamster IgG (500A2)
Armenian Hamster IgG (145-2C11)
One vial, one drop approach to compensation
OneComp eBeads® and UltraComp eBeads® are the perfect solution to compensation.
They capture mouse, rat and hamster antibodies of IgG and IgM isotypes independent
of the light chain, which means they are compatible with almost all antibodies used in
flow cytometry. Each vial of compensation eBeads contains a mixture of microspheres
that are either coated with capture antibody, resulting in a positive bead, or uncoated,
serving as a negative bead. Both beads are combined into one vial and are dispensed
as a single drop. Additionally, UltraComp eBeads have been optimized for the violet laser.
27
Figure 1C: CD4 PE is compensated for spectral overlap into the eFluor® 605NC detector using UltraComp eBeads® (left) or cells (right). Figure 2C: CD4 APC is compensated for spectral overlap into the APC- eFluor® 780 detector using UltraComp eBeads® (left) or cells (right).
Red Laser
eFlu
or® 6
05N
C
CD4 PE
eFlu
or® 6
05N
C
CD4 PE
APC
-eFl
uor®
780
CD4 APC
APC
-eFl
uor®
780
CD4 APC
Figure 1C Figure 2C
Blue Laser
PE
CD4 FITC
PE
CD4 FITC
Figure 1B: CD4 FITC is compensated for spectral overlap into the PE detector using UltraComp eBeads® (left) or cells (right). Figure 2B: CD4 PE is compensated for spectral overlap into the PerCP-eFluor® 710 detector using UltraComp eBeads® (left) or cells (right).
PerC
P-eF
luor
® 7
10CD4 PE
PerC
P-eF
luor
® 7
10
CD4 PE
Figure 1B Figure 2B
SILICA BEADS J23114 J23115_RPA-T8 605_006.fcsFSC-A, SSC-A subset9299
CD8 eFluor 605
eFlu
or 6
50
LWB_RPA-T8 605_406.fcsFSC-A, SSC-A subset10335
Comp-eFluor 605NC-A
Com
p-eF
luor
650
NC
-A
SILICA BEADS J23114 J23115_RPA-T8 605_006.fcsFSC-A, SSC-A subset9299
CD8 eFluor 605
eFlu
or 6
50
LWB_RPA-T8 605_406.fcsFSC-A, SSC-A subset10335
Comp-eFluor 605NC-A
Com
p-eF
luor
650
NC
-AeF
luor
® 6
50N
C
CD8 eFluor® 605NC
eFlu
or® 6
50N
C
CD8 eFluor® 605NC
Violet Laser
CD4 eFluor 650
APC
SILICA BEADS J23114 J23115_RPA-T8 650_008.fcsFSC-A, SSC-A subset9506
(Comp-APC-A) : Median: -10.6
(Comp-APC-A) : Median: -18.5
CD4 eFluor 650
APC
LWB_RPA-T8 650_408.fcsFSC-A, SSC-A subset10174
(Comp-APC-A) : Median: -44.8
(Comp-APC-A) : Median: -15.0
APC
eFluor® 650NC
APC
eFluor® 650NC
Figure 1A: CD8 eFluor® 605NC is compensated for spectral overlap into the eFluor® 650NC detector using UltraComp eBeads® (left) or cells (right). Figure 2A: CD4 eFluor® 650NC is compensated for spectral overlap into the APC detector using UltraComp eBeads® (left) or cells (right).
Figure 1A Figure 2A
UltraComp eBeads® suitable for all lasers
Beads Cells Beads Cells
Beads Cells Beads Cells
Beads Cells Beads Cells
28
Isotype controls �Confirm specificity of primary antibody binding
�Rule out Fc receptor mediated binding and other non-specific interactions
Selecting the appropriate isotype control is an important element in flow cytometry
experiments. Their purpose is to determine background staining and confirm specificity of the
experimental antibody. It ideally matches the host species, isotype and conjugation format, in
an effort to mimic the non-specific characteristics of the experimental antibodies used.
Isotype controls are developed to assess levels of background staining inherent in cell
binding assays. They are a good place to start when optimizing flow cytometer settings and
establishing a data range for general autofluorescence from a cell labeled with a conjugated
antibody. An isotype control antibody is expected to show low levels of staining on a
particular cell population, however sometimes during intracellular staining experiments there
may be higher levels of non-specific fluorescence.
There are several potential reasons for inconsistent staining, from inherent differences in
the amino acid composition of the two antibodies, to the different amount of fluorophore
conjugated to the isotype control versus the experimental antibody, often referred to as
fluoroscence-to-protein (F/P) ratio. Activation of cells may also alter the staining patterns
of isotype control antibodies. Therefore, using unstimulated cells is recommended, or
an inherently negative population in a heterogenous cell preparation as a more relevant
negative control, when staining intracellular targets.
Isotype Controls At-a-Glance
Description Clone
Puri
fied
Fun
ctio
nal
Gra
de
Bio
tin
eFl
uo
r® 4
50
FITC
Ale
xa F
luo
r® 4
88
Cya
nin
e5
PerC
P-C
yan
ine5
.5
PE-
eFlu
or®
610
PerC
P-eF
luo
r® 7
10
PE PE-C
yan
ine5
PE-C
yan
ine5
.5
PE-C
yan
ine7
Ale
xa F
luo
r® 5
32
APC
eFlu
or®
660
Ale
xa F
luo
r® 7
00
APC
-eFl
uo
r® 7
80
Mouse IgA n
Mouse IgM 11E10 n n n n n n
Mouse IgM eMM15 n n
Mouse IgG1 Κ P3.6.2.8.1 n n n n n n n n n n n n n n n n n n
Mouse IgG2a Κ eBM2a n n n n n n n n n n n n n n n
Mouse IgG2b Κ eBMG2b n n n n n n n n n n n n n n n n n
Mouse IgG3 n n n
Rat IgG1 Κ eBRG1 n n n n n n n n n n n n n n n n n
Rat IgG2a Κ eBR2a n n n n n n n n n n n n n n n n n n
Rat IgG2b Κ eB149/10H5 n n n n n n n n n n n n n n n n n
Rat IgM n n n n n n n n n n n
Rabbit IgG n
Armenian Hamster IgG
eBio299Arm n n n n n n n n n n n n n n n n n n
Golden Syrian Hamster IgG
n n n n n n n n n n n n
29
Secondary ReagentsSelecting the correct secondary antibody
Although directly conjugated antibodies provide a simple and robust assay detection system,
there are situations where a primary antibody followed by a secondary antibody is warranted:
�Primary antibody is not available in a conjugated format
�Primary antibody is not available in the desired format
�Amplification of primary antibody signal is needed
Understanding how to choose the appropriate secondary antibody and format is essential
for obtaining the best possible staining results.
Application determines the format
Fluorimetric (fluorochrome-conjugated) secondary antibodies are available in numerous
formats, with each specific conjugate determining the application in which the secondary
antibody can be used. The variety of available formats provides flexibility, making secondary
antibodies versatile across various assay platforms including flow cytometry, immunoassays,
immunoblotting and immunohistology.
Host species and isotype of primary determines the secondary antibody
When selecting a secondary antibody it is important to choose one that reacts with the host
species of the primary antibody. For example a Goat Anti-Mouse IgG can be used with a
mouse primary antibody. Secondary antibodies can be either polyclonal, in which case the
host species is typically goat or donkey, or monoclonal where the host species is typically
mouse or rat. Additionally, secondary antibodies are available in several classes, either IgA,
IgM or IgG, in addition to more specific subclasses such as IgG2a.
Polyclonal antibodies can provide amplification of the signal but must be highly cross-
absorbed to provide specificity to the species being stained, and must not react with the
other species or other subclasses. Most polyclonal antibodies are F(ab’)2 fragmented to
minimize Fc binding.
Monoclonal secondaries are typically used when looking at reactivity to a specific IgG
subclass. Monoclonal antibodies are consistently reactive to IgG subclasses (IgG1, IgG2a,
IgG2b, IgG2c, IgG3), in addition to providing amplification, but to a lesser extent than
polyclonal secondaries. Every eBioscience monoclonal antibody has been validated against
the specific subclass, as well as lack of reactivity to other subclasses and species. These
are useful in multiplexing when using primary antibodies from the same species
but different subclasses.
9
30
Secondary Antibodies At-a-Glance
Description Clone
Cat
. Ro
ot
Puri
fied
Bio
tin
eFlu
or®
450
FITC
PerC
P-eF
luo
r® 7
10
PE
PE-
Cya
nin
e5
PE-
Cya
nin
e7
eFlu
or®
660
APC
APC
-eFl
uo
r® 7
80
Rat Anti-Mouse IgG M1-14D12 4015 n n n n n n n n
Rat Anti-Mouse IgA 11-44-2 5994 n n
Rat Anti-Mouse IgA mA-6E1 4204 n n
Rat Anti-Mouse IgG2a m2a-15F8 4210 n n n n
Rat Anti-Mouse IgM II/41 5790 n n n n n n n n n n
Rat Anti-Mouse IgM eB121-15F9 5890 n n n n n
Mouse Anti-Rat IgM RM-7B4 4342 n
Mouse Anti-Rat IgM HIS40 0990 n n n
Mouse Anti-Rat IgG1 R1-12D10 4812 n n
Mouse Anti-Rat IgG2b R2B-7C3 4815 n n n
Mouse Anti-Rat IgG2c R2C-23A3 4816 n
Mouse Anti-Rat IgG2a r2a-21B2 4817 n n n
Donkey F(ab')2 Anti-Rat IgG Polyclonal 4822 n n
Donkey F(ab')2 Anti-Rabbit IgG
Polyclonal 4739 n
Goat F(ab')2 Anti-Mouse IgG Polyclonal 4010 n n n n n
Goat Anti-Armenian Hamster IgG
Polyclonal 4111 n
Goat Anti-Armenian Hamster IgG
Polyclonal 4112 n
Goat Anti-Golden Syrian Hamster IgG
Polyclonal 4211 n
Mouse Anti-Biotin BK-1/39 9895 n
Mouse Anti-Fluorescein isothiocyanate (FITC)
FITC-9 3300 n n n
Rat Anti-Mouse IgG Polyclonal 4013 n
Rat Anti-Mouse IgG2b m2b-25G4 4220 n
Rat Anti-Mouse IgE 23G3 5992 n n n
Rat Anti-Mouse IgD 11-26c (11-26) 5993 n n n n n n n n
Mouse Anti-Rat IgG 4811 n
Mouse Anti-Rat IgG Polyclonal 4813 n
Goat Anti-Rat IgG Polyclonal 4818
Goat Anti-Rat IgG Polyclonal 4826
Rat Anti-GFP 5F12.4 6498 n n n
Mouse Anti-GFP GF28R 6674 n
Goat F(ab')2 Anti-Rabbit IgG Polyclonal 4839 n
31
Biotin and Streptavidin Conjugates
Biotin is involved in the metabolism of fatty acids, amino acids and gluconeogenesis,
however in the laboratory, it can be used to tag molecules of interest for biochemical or
cellular studies. While Streptavidin binds to Biotin with high affinity, the fluorochrome
conjugates are commonly used with indirect staining protocols to detect biotinylated
primary antibodies in flow cytometry. The monoclonal antibody (BK-1/39) specifically
recognizes biotin and can be used as an alternative to Streptavidin.
Biotin and Streptavidin
Description Clone Application Cat. No.
Anti-Biotin Alexa-Fluor® 488 BK-1/39 FC 53-9895
Anti-Biotin PE BK-1/39 FC 12-9895
Streptavidin eFluor® 450 FC 48-4317
Streptavidin FITC FC, ICC, IHC 11-4317
Streptavidin PerCP-Cyanine5.5 FC 45-4317
Streptavidin PerCP-eFluor® 710 FC 46-4317
Streptavidin PE FC 12-4317
Streptavidin PE-Cyanine5 FC 15-4317
Streptavidin PE-Cyanine7 FC 25-4317
Streptavidin APC FC 17-4317
Streptavidin eFluor® 660 FC, ICC, IHC 50-4317
Streptavidin APC-eFluor® 780 FC 47-4317
Streptavidin Cyanine5 FC 19-4317
Streptavidin eFluor® 710 FC 49-4317
32
Instrument and fluorochrome/dye chart
EXCITATIONLASER*
VIOLET (405 nm) BLUE (488 nm)RED (633-647 nm)
UV (325-355 nm)GREEN (532 nm)
YELLOW (561-570 nm)
DYES
Emission Max (nm)
Calce
in Bl
ue A
M (4
45)
FVD
eFlu
or® 4
55UV
Calce
in V
iole
t AM
(450
) C
PD eF
luor
® 450
FVD
eFlu
or® 4
50
FVD
eFlu
or® 5
06
CSD
eFlu
or® 5
14
CF
SE (5
21)
Calce
in A
M (5
15)
FVD
eFlu
or® 5
20
PI (6
17)
7-AA
D (6
47)
FVD
eFlu
or® 6
60
CPD
eFlu
or® 6
70
FVD
eFlu
or® 7
80
FLUOROCHROME
Emission Max (nm)
eFlu
or® 6
05NC
eFlu
or® 6
25NC
eFlu
or® 6
50NC
eFlu
or® 4
50
Alex
a Flu
or® 4
88 (5
19)
FITC (
520)
PerC
P (67
8)
PerC
P-Cy
anin
e5.5
(695
)
PerC
P-eF
luor
® 710
PE (5
78)
PE -e
Fluor
® 610
PE-C
yani
ne5 (
667)
PE-C
yani
ne5.
5 (69
5)
PE-C
yani
ne7 (
785)
Alex
a Flu
or® 5
32 (5
61)
APC (
660)
Cyan
ine5
(6
70) e
Fluor
® 660
eFlu
or® 7
10
Alex
a Flu
or® 7
00 (7
23)
APC-
eFlu
or® 7
80
BANDPASS FILTERS*
450/50 605/40 660/40 450/50 510/50 530/30 575/26 670/14 695/40 710/50 575/26 610/20 670/14 695/40 780/60 560/14 660/20 710/50 780/60
FACSCalibur (2-laser), Accuri C6
FL1 FL2 FL3 FL2 FL3 FL3 FL4
FC500, FACSCanto, EasyCyte 8HT
FL1 / Green
FL2 / Yellow FL4 / Red 1 FL2 /
Yellow Red 1 FL4 / Red 1 FL5/Infra Red 1
FL4 / Red 2
FL5/Infra Red 2
MACSQuant FL1 FL1 FL2 FL3 FL4 FL3 Red 1 FL4 FL5 FL6 FL7
Gallios Blue Blue Yellow 2 Green Yellow 1 Red 1 Yellow 1 FL4 Red 1 Infra Red 1 Red 2 Far Red Infra
Red 2
FACSCanto II (3-laser)
FACSVerse (4-2-2)Blue Blue Green /
Green2Green / Green 1 Yellow Red 1 Yellow Red 1 Red 1 Infra
Red 1 Red 2 Infra Red 2
CyAn ADP FL6 FL7* FL7* FL6 FL7* FL1 FL2 FL4 FL2 Red 1 FL4 FL5 FL8 FL9
LSR II (4-2-2) FL5/7 FL6* FL6* FL5 FL6* FL1 FL2 FL3 FL2 Red 1 FL3 FL4 * FL10 FL11
iCyt Eclipse (4-laser)
FL1 FL3 FL1 FL2 FL3 FL4* FL3 FL4 FL4* FL5 FL4* FL5
Note: Peak emission for eFluor® dyes is noted in the name. Peak emission for all other dyes is shown in parentheses. Before combining reagents in multicolor experiments, always refer to your specific system configuration. *Available lasers, bandpass and longpass filters will vary depending on the configuration.
Key: CSD = Calcium Sensor Dye; CPD = Cell Proliferation Dye; FVD = Fixable Viability Dye; PI = Propidium Iodide
All trademarks used are the property of their respective owners. Customers in countries where direct sales are not available may contact their eBioscience distributor listed at www.eBioscience.com/distributors.
SPPT02821-1 Beads, Buffers, Dyes 1113
Customers in countries where direct sales are not available may contact their eBioscience distributor listed at www.eBioscience.com/distributors
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